PROJECT SUMMARY Insulin and insulin-like growth factor-1 (IGF1) are closely related polypeptide hormones/growth factors that regulate cell growth and metabolism. Insulin and IGF1 exert their biological effects on target cells by binding to distinct cell surface receptors, the insulin receptor (IR) and the IGF1 receptor (IGF1R), respectively, which are structurally related receptor tyrosine kinases. IR and IGF1R have low basal activity, which is stimulated through ligand binding to the ectodomains, resulting in autophosphorylation of specific tyrosine residues in the cytoplasmic tyrosine kinase domains. Aberrant signaling by IR and IGF1R is relevant to human disease: decreased signaling of IR plays a role in non-insulin-dependent diabetes mellitus, while increased signaling of IGF1R and IR is implicated in cancer. Despite extensive structural and biochemical studies of the separate ectodomains and cytoplasmic domains of these receptors, a comprehensive understanding of the signal transduction mechanism for IR and IGF1R is still lacking, in large part due to the paucity of structural information for the holoreceptors. To address these issues, we have produced highly purified preparations of IR and IGF1R that are detergent-soluble, functional, and responsive to their ligands. We propose the first detailed kinetic analyses of IR and IGF1R holoreceptors in purified form. We will also use single-particle electron microscopy to determine the structures of the holoreceptors. Finally, we will use biochemical approaches and crystal structures of the kinase domains to understand the relative positioning of the tyrosine kinase domains in the unliganded, basal and phosphorylated, activated states. We will also test the hypothesis that the C-terminal tails of IR and IGF1R are involved in enzyme regulation. These structure-function studies will be correlated with studies of the metabolic functions of IR and the growth-promoting effects of IGF1R in cells lacking the receptors. The work proposed in this grant application will advance our knowledge of the molecular mechanisms involved in transmembrane signaling by IR and IGF1R. This information should prove to be valuable in the design of small-molecule agonists that act intracellularly to activate IR; such agonists would have potential use as anti-diabetic therapeutics. In addition, from an understanding of the activation mechanism, it might be possible to develop IR/IGF1R inhibitors to block receptor signaling in cancer cells.